Studies of a novel 3-0 sulfatase specific for a unique glucosamine residue found within a short antithrombin binding sequence of heparin have been extended. The enzyme has been demonstrated in plasma and in human leukocytes and has been partially purified from human urine. The enzyme has a molecular weight of approximately 65,000 and manifests three isoelectric points extending from pH 5.5 and 4.5. A heat-stable, macromolecular inhibitor is present in urine and coprecipates with the enzyme in the presence of ammonium sulfate. The unique sulfated glucosamine residue is located near the non-reducing terminus of the heparin chain and this novel sulfatase may be essential for the metabolic degradation of heparin. The absence of this enzyme would lead to a variant of the Sanfilippo syndrome. To establish the specificity of the enzyme and to help identify the glucosamine sulfate residues found in heparin, a number of isomeric O-sulfated glucosamine derivatives have been prepared. These may serve as standards for establishing the structure of sulfated hexosamine residues found in oligosaccharides linked to anterior pituitary hormones and other glycoproteins.